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Published ahead of print on April 21, 2005, doi:10.1165/rcmb.2003-0341OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 121-129, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2003-0341OC

Cigarette Smoke Extract Induces DNA Damage but Not Apoptosis in Human Bronchial Epithelial Cells

Xiangde Liu, Heather Conner, Tetsu Kobayashi, Huijung Kim, Fuqiang Wen, Shinji Abe, Qiuhong Fang, Xingqi Wang, Mitsuyoshi Hashimoto, Peter Bitterman and Stephen I. Rennard

University of Nebraska Medical Center, Omaha, Nebraska; University of Minnesota, Minneapolis, Minnesota; and Seoul Adventist Hospital and WonKwang University Kunpo Medical Center, Seoul, Republic of Korea

Correspondence and requests for reprints should be addressed to Stephen I. Rennard, M.D., University of Nebraska Medical Center, 985885 Nebraska Medical Center, Omaha, NE 68198-5885. E-mail: srennard{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Whether DNA damage caused by cigarette smoke leads to repair or apoptosis has not been fully elucidated. The current study demonstrates that cigarette smoke induces single-strand DNA damage in human bronchial epithelial cells. Cigarette smoke also stimulated caspase 3 precursors as well as intact poly (ADP-ribose) polymerase (PARP) production, but did not activate caspase 3 or cleave PARP, while the alkaloid camptothecin did so. Neither apoptosis nor necrosis was induced by cigarette smoke when the insult was removed within a designated time period. In contrast, DNA damage following cigarette smoke exposure was repaired as evidenced by decreasing terminal dUTP-biotin nick-end labeling positivity. The PARP inhibitor, 3-aminobenzamide blocked this repair. Furthermore, cells subjected to DNA damage were able to survive and proliferate clonogenically when changed to smoke-free conditions. These results suggest that cigarette smoke–induced DNA damage in bronchial epithelial cells is not necessarily lethal, and that PARP functions in the repair process. Our data also suggest that the potency of cigarettes as a carcinogen may result from their ability to induce DNA damage while failing to trigger the apoptotic progression permitting survival of cells harboring potentially oncogenic mutations.

Key Words: apoptosis • DNA content • PARP • TUNEL


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Cigarette smoke contains more than 6,000 components, many of which can lead to DNA damage (1). Consistent with this, cigarette smoke exposure has been reported to induce DNA damage in a variety of cell types (2, 3). Clinically, one of the major cell types affected by cigarette smoke is the airway epithelium, where damage can lead to cancer and may contribute to the development of chronic obstructive pulmonary disease (4–6).

When damage occurs to the DNA of a cell, several responses are possible. Often apoptosis or programmed cell death occurs, a response thought to protect the integrity of the genome. Apoptosis is a naturally occurring biological process associated with a wide variety of fundamental life systems, including cell development, differentiation, and response to injury. It can be activated by at least two major pathways either in response to activation of so-called "suicide" receptors, or in response to cellular damage that leads to mitochondrial release of cytochrome C (7). Both pathways activate common effector mechanisms involving a cascade of intracellular proteases, the caspases, and disrupt DNA repair mechanisms by cleaving repair enzymes such as poly (ADP-ribose) polymerase (PARP) (8). PARP has been suggested to play a key role in determining whether DNA injury leads to repair or to apoptosis (9).

Apoptosis of lung epithelial cells could be protective if it prevents the survival of cells with altered genetic programming. Alternatively, excessive apoptosis could lead to a loss of lung cells and could contribute to diseases such as emphysema. The present study, therefore, assessed the ability of cigarette smoke to induce DNA damage in cultured airway epithelial cells and determine whether that damage induces apoptosis or repair. Using the terminal dUTP-biotin nick-end labeling (TUNEL) assay, which detects DNA strand breaks, DNA damage in response to cigarette smoke was demonstrated. This damage, however, did not lead to apoptosis and, moreover, was reversible, a process which depended on the activity of the DNA repair enzyme PARP.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials and Cell Culture
LHC basal medium was purchased from Biosource International (Camarillo, CA). RPMI 1640 medium, trypsin/EDTA, penicillin G sodium, and streptomycin were purchased from Invitrogen (Life Technologies, Grand Island, NY) and amphotericin B from Pharma-Tek (Elmira, NY). The TiterTACS Colorimetric Apoptosis Detection Kit was purchased from Trevigen (Gaithersburg, MD). The cell death detection kit (TUNEL assay) was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Goat anti–caspase 3 antibody (CPP32), which reacts with both precursor and active forms of human caspase 3, and goat anti-PARP, which reacts with both intact and cleaved forms of human PARP, were purchased from R&D Systems (Minneapolis, MN). Rabbit source anti-goat IgG horseradish peroxidase was purchased from Rockland Immunochemicals (Gilbertsville, PA). Propidium iodide, camptothecin (CPT), and 3-aminobenzamide (3-ABA) were purchased from Sigma (St. Louis, MO), and Vitrogen 100 was purchased from Cohesion Technologies (Palo Alto, CA).

The human bronchial epithelial cell line, BEAS-2B, was obtained from the American Type Culture Collection (#CRL-9609; Rockville, MD). Cells were cultured in 100 mm collagen (Vitrogen 100)-coated tissue culture dishes (Falcon; BD Bioscience Discovery Labware, Bedford, MA) in a 1:1 mixture of LHC-9/RPMI 1640 (10). The cells were fed 2–3 times a week. Confluent cells were detached by 0.05% trypsin in 0.53 mM EDTA and suspended in LHC-9/RPMI containing soybean trypsin inhibitor.

Normal human bronchial epithelial cells (HBECs) were acquired from bronchial biopsies using a previously published method with modifications (11). HBECs were cultured under serum-free conditions using a 1:1 mixture of LHC-9/RPMI 1640. Cells were plated on collagen (Vitrogen 100)-coated tissue culture dishes at 37°C in a humidified, 5% CO2 atmosphere. Cells were passaged once a week at a 1:3 ratio. Cells between the 3rd and 10th passage were used for experiments.

Cigarette Smoke Extract Preparation
Cigarette smoke extract (CSE) was prepared with a modification of the method of Carp and Janoff (12). Briefly, one 100-mm cigarette without filter (Research Grade Cigarette, University of Kentucky) was combusted with a Variable Speed Pump (Fisher Scientific, Pittsburgh, PA). The smoke was bubbled through 25 ml double-distilled water (ddH2O) at a speed of 50 cc/min. The resulting suspension was filtered through a 0.22-µm pore filter (Lida Manufacturing Corp., Kenosha, WI) to remove bacteria and large particles. This solution was considered to be 100% CSE and diluted with LHC-D/RPMI 1640 medium (10) within 30 min of preparation to obtain the desired concentration in each experiment.

TUNEL Staining
DNA damage was evaluated and quantified with a colorimetric apoptosis detection kit (Titer TACS; Trevigen) that uses TUNEL stain in a 96-well format, following the manufacturer's instructions. Briefly, cells were cultured in 12-well plates till confluent and treated with CSE. Cells were then trypsinized and suspended in LHC-D/RPMI containing STI. The cells were counted and transferred into a round-bottom 96-well plate (2 x 105cells/well). Cells were then fixed with 3.7% buffered formaldehyde for 5 min followed by washing once with phosphate-buffered saline (PBS). Cells were permeabilized with 100% methanol for 20 min followed by washing once with PBS. Cells were then subjected to labeling procedure following the manufacturer's instructions. The absorbance was measured at 450 nm with Benchmark microplate reader (Bio-Rad, Hercules, CA). For comparison, data were expressed as percentage of Control at time 0, which is (Sample OD value-blank)/(Control OD value at time 0 – blank) x 100%.

In addition, cells were assessed histochemically using the fluorescence-labeled TUNEL assay kit (Roche Molecular Biochemicals). Briefly, cells were cultured in V30-coated 8-chamber slides till subconfluent. Cells were treated with CSE and then fixed with freshly prepared paraformaldehyde (4% in PBS; pH 7.4) for 1 h at room temperature. The cells were permeabilized with 0.1% Triton X-100 (in 0.1% sodium citrate) for 2 min at 4°C and rinsed with the PBS. The cells were then reacted with the TUNEL mixture in a humidified chamber for 60 min at 37°C in the dark. After the cells were washed three times with PBS, they were counterstained with 1 µg/ml of propidium iodide in a humidified chamber for 20 min at 37°C in the dark. After washing with PBS, the fluorescent incorporation into nucleotide was detected under fluorescent microscopy at x400 magnification.

Detection of Cell Viability
Cell viability was evaluated by 3-(4,5-dimenthylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay using previously described methods (13). Briefly, cells were cultured in 12-well plates in parallel with the samples for TiterTACS assay. After treatment with CSE, cells were incubated with MTT solution (0.5 mg/ml in LHC-D/RPMI) for 4 h. Cells were washed once with PBS. Formazan crystals were then dissolved with DMSO by shaking for 1 h at room temperature. Absorbance was then observed with a Benchmark microplate reader (Bio-Rad) using a 540-nm wavelength filter. Absolute optical density was obtained and expressed as percent of control.

DNA Strand Break Assay
DNA strand break (DNA-SB) assay was performed as described in the published reports (14, 15). Briefly, confluent HBECs were exposed to CSE or 1 µM CPT for 6 h. Cells were also treated with H2O2 (100 µM) for 30 min as a positive control for DNA strand breaks. After washing once with PBS, cells were trypsinized, pelletted, and resuspended with 300 µl of solution B (0.25 M Mesoinositol, 10 mM sodium phosphate, 1 mM MgCl2, pH 7.2) to give a cell density ~ 10 x 106 cells/ml. Aliquots of this suspension were then distributed into three Eppendorf tubes (100 µl each), designated T, P, and B. After that, 100 µl of solution C (9 M urea, 10 mM NaOH, 2.5 M cyclohexanediamineacetate, and 0.1% sodium dodecyl sulfate) was added without mixing and incubated at 0°C for 10 min to enable cell lysis and chromatin disrupture. To the tubes P and B, 50 µl of solution D (0.45 vol of solution C in 0.2N NaOH) and 50 µl of solution E (0.4 vol of solution C in 2 N NaOH) were added gently without mixing followed by sonication of the tube B for 3 s to ensure rapid denaturation of the DNA in the alkaline solution. All tubes were then incubated at 0°C for 30 min. After incubation, 200 µl of solution F (1 M glucose and 14 mM mercaptoethanol) were added to tubes T, P, and B followed by adding solutions D and E into T tubes. After sonication of tubes T and P, 750 µl of solution G (6.7 µg/ml ethidium bromide in 13.3 mM NaOH) was added to all the tubes. Fluorescence was then determined using a Spectra MAX Gemini Fluorometer (Molecular Devices, Sunnyvale, CA) with excitation at 520 nm and emission at 585 nm. Results are expressed as % ds-DNA = (P – B)/(T – B) x 100, where P is the fluorescence of the experimental condition, B is the background ethidium bromide fluorescence determined after converting all the DNA into single-strand form, and T is the fluorescence determined after adding the mercaptoethanol solution before the alkaline solution.

Comet Assay
Comet assay was performed using CometAssay Kit (Trevigen, Inc.). Briefly, cells were cultured in 6-well plates and treated with CSE or CPT for 6 h. Cells were harvested by trypsinizing and combined with floating cells in the medium containing STI. The cells were pelleted and resuspended with cold PBS at 105 cells/ml. Fifty microliters of the cell suspension was mixed with 500 µl of LMAgarose, and 75 µl of the agarose/cells was pipetted over sample area of CometSlides. The slides were placed flat at 4°C humidity chamber for 30 min, after which they were immersed in cold Lysis Solution and left at 4°C overnight. The slides were then transferred into Alkali Solution and incubated at room temperature for 60 min by changing the Alkali Solution once. The slides were then horizontally placed in an electrophoresis apparatus and run at 1 Volt/cm for 15 min. After fixing with 70% ethanol for 5 min, slides were air-dried and stained with SYBR. Cells were then viewed with an epifluorescence microscopy (Nikon Eclipse E800; Nikon, Melville, NY) and photographed with a digital camera (Optronics, Goleta, CA) under x200 magnification. The measurement was performed using a public domain PC-image analysis program CASP software (16). The following comet parameters were analyzed using the software: head length (Lhead); tail length (Ltail); comet length (Lcomet); head DNA; tail DNA; tail moment (TM); and olive tail moment (OTM). In addition, apoptotic index was calculated as the percent of cells with diffuse fan-like tail and very small heads from a minimum of three slides counted per condition (17).

Profile of DNA Content by Flow Cytometry
To determine the presence of apoptotic cells, DNA content was measured by flow cytometry as reported previously (18). Cells were cultured in 6-well plates till confluent. After treatment with CSE or as a positive control, the DNA topoisomerase inhibitor CPT, medium was harvested to collect floating cells and attached cells were detached from the tissue culture dishes with trypsin/EDTA. Cells were then pelleted together and fixed with 70% ethanol at 4°C for 30 min. After staining with propidium iodide (50 µg/106 cells), cell cycle analysis was performed by flow cytometry. Cells with less DNA staining than that of G1 cells (sub-G1 peaks or A0 cells) were considered apoptotic.

Measurement of Caspase 3 Activity
Caspase 3 activity was measured with a commercially available colorimetric kit from R&D Systems, following manufacturer's instructions. Briefly, cells were cultured in 60-mm dishes and treated with CSE or CPT for 6 h. Both floating and attached cells were harvested. Cell lysis buffer (provided by the vendor, R&D Systems, 200 µl/sample) was added into the cell pellets and caspase-3 activity was measured.

Immunoblots for Caspase 3 and PARP
Cells were lysed with lysing buffer (50 mM Tris buffer, pH 7.4, containing 10 mM EDTA, 2 mM EGTA, 2 mM benzamidine, 2.5 mM dithiothreitol, 2 µg/ml soybean trypsin inhibitor, 100 µM tosyl-L-lysine chloromethyl ketone, 200 µM leupeptin, and 50 µM phenylmethylsulphonyl fluoride). The cell lysate was centrifuged at 12,000 x g for 10 min at 4°C and the precipitates were discarded. Protein concentrations in supernatants were determined by a protein dye–binding assay (Bio-Rad). Proteins were then subjected to immunoblot analysis. After heating for 3 min at 95°C, 10 µg of total protein was mixed with 2x sample buffer (0.5 M Tris-HCL, pH 6.8, 10% SDS, 0.1% bromphenol blue, 20% glycerol, 2% ß-mercaptoethanol) and loaded into each well before performing electrophoresis with the Mini-protein 3 Cell System (Bio-Rad). The proteins were transferred to PVDF membranes (Bio-Rad) in transfer buffer (20 mM Tris, pH 8.0, 150 mM glycine, 20% methanol) at 20 V for 40 min with the semi-dry electrophoretic transfer system (Bio-Rad). The membrane was blocked with 5% nonfat milk in PBS-Tween at room temperature for 1 h and then exposed to primary antibodies (R&D Systems) at 4°C overnight. Target proteins were subsequently detected using rabbit anti-goat IgG horseradish peroxidase (Rockland, Gilbertsville, PA) in conjunction with an enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK).

Clonogenicity Assay
Clonogenicity assay was performed with a modification of the previously reported methods (19). Briefly, cells were cultured in 60-mm dishes and treated with CSE or CPT for 6 h. Both floating and attached cells were harvested. After counting the number, cells were then plated in noncoated 60-mm tissue culture dishes at 2 x 103 cells/ml, 5 ml/dish in LHC-9/RPMI. Cells were maintained in culture for 7–10 d, with the medium being changed every 3–4 d. Cells were then fixed with PROTOCOL (Fisher Diagnostics, Middletown, VA) and photographed. Colonies, defined as cluster of 20 or more cells, were scored under an inverted microscope.

Statistical Analysis
All quantitative data were expressed as means ± SEM determinates from representative experiments. Each experiment was repeated at least three times, unless otherwise indicated, with similar results. Comparison of paired data was performed using the Student t test, whereas multigroup data was analyzed by ANOVA followed by Tukey correction. P < 0.05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effect of Cigarette Smoke on DNA Damage and Cell Viability
To study the effect of cigarette smoke on DNA damage, bronchial epithelial cells were exposed to either 5% CSE or 1 µM CPT for 24 h. The quantitative in situ TUNEL assay, in parallel with the MTT assay, was performed after 1-, 2-, 4-, 6-, and 24-h exposure. As shown in Figure 1, even 1 h of exposure to 5% CSE or CPT caused a significant increase of TUNEL-positive cells (54.9 ± 3.0% by 5% CSE and 31.2 ± 2.2% by CPT, respectively, P < 0.05 compared with control of 16.2 ± 0.3%, Figure 1A). TUNEL-positive cells gradually increased to nearly 90% in both 5% CSE- and CPT-treated groups as a function of time, although the 5% CSE effect was stronger than 1 µM CPT during the first 4 h. Cell viability as assessed by MTT, however, was not affected by 5% CSE but was significantly reduced by CPT at 6 h (100.3 ± 3.3% versus 61.3 ± 5.5% of control, P < 0.05) and at 24 h (103.2 ± 7.6% versus 26.6 ± 1.9% of control, P < 0.05; Figure 1B).




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  		Figure 1. Effect of CSE on DNA damage and viability in BEAS-2B cells: time course. (A) DNA damage, TUNEL assay. Confluent BEAS-2B cells were treated with (triangles) or without 5% CSE or 1 µM CPT (diamonds) for up to 24 h, and TiterTACS TUNEL assay was performed at each time point indicated. Horizontal axis: time (h); vertical axis: percent of TUNEL-positive cells versus DNase-treated control. Squares, LHC-D/RPMI. (B) Cell viability, MTT assay. In parallel with TiterTACS assay, cell viability was tested by MTT assay. Horizontal axis: time (h); vertical axis: percent of viable cells versus control at 0. *P < 0.05 versus control at respective time points. Shown is one representative experiment of three, each measured in duplicate.

 
Cigarette smoke–induced DNA damage was dependent on CSE concentration. After 6 h exposure to increasing concentrations of CSE, the number of TUNEL-positive cells dramatically increased without affecting cell viability (24.4 ± 8.3%, 50.1 ± 6.7% and 85.6 ± 6.3%, respectively, Figure 2A). Similar results were found with normal human bronchial epithelial cells (Figure 2B).




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  		Figure 2. Effect of CSE on DNA damage and cell viability in epithelial cells: concentration dependence. Confluent BEAS-2B or HBECs were treated with varying concentrations of cigarette smoke for 6 h. TiterTACS TUNEL assay (squares) in parallel with MTT assay (triangles) were then performed. (A) BEAS-2B cells. Left vertical axis: percent of TUNEL-positive cells versus DNase-treated control; right vertical axis: cell viability. Horizontal axis: CSE concentration. (B) HBEC. Vertical axis: percent of TUNEL-positive cells versus DNase-treated cells. Horizontal axis: CSE concentration. *P < 0.05 compared with 0% CSE. Shown is one representative experiment of three, all with similar results.

 
To further study if CSE induced single-strand (ss)-DNA damage or double-strand (ds)-DNA damage, DNA-SB was assessed by alkaline unwinding and ethidium bromide fluorescence binding method. As shown in Figure 3, CSE (2.5, 5, and 10%) caused negligible alterations in HBECs ds-DNA after 6 h exposure, whereas CPT significantly decreased ds-DNA after 4 h treatment (P = 0.02). As a positive control, H2O2 very significantly reduced ds-DNA (Figure 3).



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  		Figure 3. DNA-SB assay. Confluent HBECs were treated with varying concentrations of CSE or 1 µM CPT for 6 h. For positive control, cells were also treated with 100 µM H2O2 for 30 min. Cells were harvested and DNA-SB assay was performed as described in MATERIALS AND METHODS. *P = 0.02 compared with control.

 
Effect of Cigarette Smoke on DNA Content and Alkaline Comet Assay
To determine whether CSE-induced DNA-damage led to apoptosis, the DNA content of populated cells was analyzed by flow cytometry, and DNA damage in individual cells was evaluated by Comet assay. After 24 h exposure to 5% CSE, the DNA content of cells did not change (Figure 4A, middle row). In contrast, sub-G1 peaks (green) gradually increased as a function of time in 1 µM CPT–treated cells (Figure 4A, bottom row), indicating that apoptosis occurred in these cells. Moreover, although CSE showed a clear concentration-dependent increase in TUNEL positivity, no effect was observed on the number of cells with hypodiploid DNA content with CSE concentrations of up to 10% in either HBECs or BEAS-2B cells (Figures 4B and 4C).





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  		Figure 4. Effect of CSE on DNA content by flow cytometry. (A) Time dependence. Confluent BEAS-2B cells were treated with or without 5% CSE or 1 µM CPT up to 24 h. Cells were harvested at the indicated times and DNA content was analyzed by flow cytometry. (B and C) CSE concentration–dependent effect in HBECs (B) and BEAS-2B (C). Confluent HBEC or BEAS-2B cells were treated with varying concentrations of CSE or 1 µM CPT for 6 h. Cells were then harvested and DNA content was analyzed by flow cytometry. Vertical axis: number of cells; horizontal axis: channel number corresponding to DNA content. Left red peak: cells with 2N DNA content corresponding to G0, G1 phases; right red peak: cells with 4N DNA content corresponding to G2, M phases; hatched area: cells corresponding to S phase; green peak: cells with less than 2N DNA scored as apoptotic. Shown is a representative of two separate experiments, both with similar results.

 
By single-cell electrophoresis assay, also known as Comet assay, we evaluated DNA damage and apoptosis using a public domain software (CASP) as described previously (16). Although there were no differences in tail length, head length, head DNA, or tail DNA between control and 5% CSE–treated cells, the Lcomet, TM, and OTM were significantly higher in 5% CSE–treated cells compared with control cells (P < 0.05, Table 1), indicating that cigarette smoke caused DNA strand(s) breaks. In addition, the Apoptotic Index (AI), that is, the percent of cells with diffuse fan-like tail and small head, was calculated by counting the cells because these cells were not suitable to analyze with the CASP software. A large proportion of CPT-treated cells (AI: 41.5 ± 4.0%), whereas only a few of 5% CSE–treated (AI: 4.0 ± 3.3%) or control cells (AI: 5.6 ± 2.7%) had diffuse fan-like tail with very small head (Figure 5 and Table 1), indicating that cigarette smoke did not increase apoptosis whereas CPT did.


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  		TABLE 1. Analysis of comet assay*

 


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  		Figure 5. Comet assay. Confluent HBECs were treated with 5% CSE or 1 µM CPT for 6 h. Both floating and attached cells were then harvested, mixed with LMAgarose, and placed in CometSlides. Cells were treated with lysis buffer overnight and subjected to electrophoresis horizontally. After fixing and staining, cells were observed and photographed under fluorescence microscopy with x200 magnification. Comet images were analyzed using CASP software as described in MATERIALS AND METHODS.

 
Effect of Cigarette Smoke on Caspase 3
To explore the role of caspase 3 on CSE-induced DNA damage, immunoblotting for caspase 3 and functional evaluation of caspase 3 activity was performed. Under control conditions, BEAS-2B cells produced the 32-kD caspase 3 precursor, and cigarette smoke significantly increased its steady state level in the cell (Figure 6A). In accord with the apoptosis data, cigarette smoke did not convert the precursor form of caspase 3 (32 kD) into the active form (18 kD or smaller fragments), whereas CPT did (Figure 6A). Consistently, no difference in caspase 3 activity between CSE-treated cells and control cells was observed (Figure 6B).




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  		Figure 6. Effect of CSE on caspase 3 synthesis and activation. Subconfluent BEAS-2B cells were treated with 1 µM CPT or indicated concentrations of CSE for 6 h. (A) Immunoblot for caspase 3 and ß-actin. (B) Caspase 3 activity. Vertical axis: percent of caspase 3 activity versus CPT-treated cells. Horizontal axis: CSE concentrations. Shown is one representative experiment of three, all with similar results.

 
Reversibility of CSE-Induced DNA Damage
In response to CSE-induced DNA damage, cells may initiate a series of repair processes by activating DNA repair enzymes, such as PARP. To evaluate this, cells were treated with 5% CSE for 6 h, then allowed to recover from the damage in the presence of growth factors in smoke-free medium. Both qualitative (fluorescence staining) and quantitative (TiterTACS) TUNEL assays were performed during the recovery period. After exposure to 5% CSE for 6 h, the majority of cells became TUNEL-positive. However, TUNEL positivity gradually decreased as a function of recovery time. After 48 h, no difference in TUNEL positivity between the CSE-treated cells and control cells was observed (Figure 7A), indicating that DNA breaks detected by TUNEL were eliminated during this period. Similar results were found by the quantitative TiterTACS method (Figure 7B).




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  		Figure 7. Reversibility of CSE-induced DNA damage. (A) TUNEL Staining. Subconfluent BEAS-2B cells were treated with (triangles) and without 5% CSE (squares) for 6 h. Cells were continuously cultured with (inverted triangles) or without (triangles) CSE in LHC-9/RPMI for 48 h. TUNEL staining was performed at indicated time point. Propidium iodide (PI) only and DNase I were used as negative and positive controls. Shown is one example of two independent experiments, both with similar results.

 
To further determine whether CSE-induced DNA damage is repairable and cells are viable, clonogenicity assay was performed immediately after CSE exposure. As shown in Figure 8, clonogenic surviving fraction in CSE-exposed cells was similar or higher (118.5 ± 8.2 colonies of 2.5% CSE–treated cells versus 89.7 ± 6.8 of control cells, P < 0.05) to that of control cells, whereas CPT-treated cells significantly lost surviving ability (89.7 ± 6.8 colonies of control versus 6.3 ± 1.3 of CPT-treated cells, P < 0.01).




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  		Figure 8. Effect of cigarette smoke on clonogenic survival of HBECs. Confluent HBECs were treated with 1 µM CPT or varying concentrations of CSE as indicated. Both floating and attached cells were harvested and immediately plated in 60-mm tissue culture plates without coating (2 x 103 cells/ml, 5 ml/dish). After 8 d of culture, cells were fixed with PROTOCOL and photographed. (A) One representative of six different experiments in both HBEC and BEAS-2B cells. (B) Quantification of colony formation. Colonies of 20 or more cells were scored under inverted microscope after 7–10 d. Six dishes per condition were counted.

 
The Role of PARP in DNA Repair
To evaluate whether the DNA repair enzyme PARP played a role in recovery following smoke-induced DNA damage, two experiments were performed. First, smoke-induced activation of PARP was assessed by Western blot. Second, PARP activity was blocked using the inhibitor 3-aminobenzamide (3-ABA). Cigarette smoke stimulated intact PARP (116 kD) protein synthesis in a concentration-dependent manner, whereas CPT decreased intact PARP by cleaving it into a smaller molecular mass (23 kD; Figure 9). In contrast to the repair observed when smoke-exposed cells were allowed to recover in control medium, in the presence of 3-ABA, DNA repair was completely blocked (Figure 10). Moreover, in the presence or absence of 5% CSE, 3-ABA (1 mM) did not induce apoptosis, as demonstrated by DNA content profiling (Figure 11).



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  		Figure 9. Immunoblots for PARP. Cells were treated with 1 µM CPT or indicated concentrations of CSE for 6 h. Cell lysates were then immunoblotted for PARP (both intact and cleaved forms). After stripping, the membrane was blotted with anti–ß-actin antibody. Shown is a representative of three separate experiments, all with similar results.

 


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  		Figure 10. Effect of PARP inhibition on recovery from DNA damage induced by CSE. Confluent BEAS-2B cells were treated with 5% CSE with or without 1 µM 3-ABA for 6 h. Cells were then cultured for 3 d in LHC-9/RPMI. Vertical axis: percent of TUNEL-positive cells versus DNase-treated cells; horizontal axis: time (h). Shown is a representative of three separate experiments, all with similar results. Filled squares, control; triangles, 3-ABA (1 mM); open squares, 5% CSE for 6 h; diamonds, 5% CSE for 6 h + ABA; circles, 5% CSE for 72 h.

 


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  		Figure 11. Effect of 3-ABA on DNA content in the presence of CSE. Confluent HBECs were treated with 5% CSE and/or 1 mM 3-ABA for 24 h. Cells were then harvested and DNA content was analyzed by flow cytometry. For each panel, the vertical axis is the number of cells, and the horizontal axis is the channel number corresponding to DNA content. Left red peak: cells with 2N DNA content corresponding to G0, G1 phases; right red peak: cells with 4N DNA content corresponding to G2, M phases; hatched area: cells corresponding to S phase; green peak: cells with less than 2N DNA scored as apoptotic. Shown is a representative of two separate experiments, both with similar results.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The current study, using TUNEL and Comet assays, demonstrates that cigarette smoke induces DNA damage in "normal" HBECs, as well as BEAS-2B cells. This effect was dependent on the duration of smoke exposure and the concentration of smoke. When the smoke was removed, however, cells did not undergo apoptosis, as assessed by flow cytometry of DNA content and Comet assay, or necrotic death, as assessed by MTT assay. In contrast, CPT, a DNA topoisomerase inhibitor, induced not only DNA damage but also apoptosis in these cells. CPT also increased caspase 3 activity, whereas cigarette smoke did not activate caspase 3, as revealed by functional activity and immunoblot, although smoke increased synthesis of the caspase 3 precursor in a concentration-dependent manner. In addition, cigarette smoke–induced DNA damage was reversible as evidenced by a time-dependent decrease in TUNEL positivity and clonogenic survival of the cells. Smoke stimulated intact PARP protein expression in a concentration-dependent manner. The PARP inhibitor, 3-ABA, significantly blocked DNA repair, as revealed by unchanged TUNEL positivity after removal of CSE, but apoptosis was not initiated.

Several studies have reported that cigarette smoke can induce necrosis or apoptosis in a variety of cells, including an alveolar epithelial cell line (A549) and human fetal lung fibroblasts (3, 20). It also has been reported that cigarette smoke causes DNA damage in A549 cells by activating endonuclease (21), and that cigarette smoke augmented asbestos-induced alveolar epithelial cell injury through a free radical–dependent mechanism (22). The current study extends these findings by demonstrating that cigarette smoke induces ss-DNA damage as evidenced by positive TUNEL assay and increased TM by Comet assay, but negative DNA-SB assay in human bronchial epithelial cells as well as the BEAS-2B cell line, and that this effect is dependent on the duration and concentration of cigarette smoke exposure. Interestingly, however, in the current study neither apoptosis nor necrosis occurred after DNA damage.

In response to DNA damage, multiple cellular processes, including cell cycle checkpoint activation, DNA repair, and apoptosis are initiated to maintain genomic integrity. Either under- or overactivation of these biological processes results in genomic instability, DNA mutation, phenotypic transformation, and functional alteration. The current study demonstrates that in addition to causing DNA damage, cigarette smoke permits DNA repair processes to occur. Whether the cells undergo apoptotic death or survive the injury after DNA damage may largely depend on the mechanism of DNA damage and repair. In this regard, the current study demonstrates that cigarette smoke–induced DNA damage in bronchial epithelial cells is not lethal, with cells surviving damage, as evidenced by a decrease in TUNEL-positive cells to the level of control after 2 or 3 d after the smoke is removed. Furthermore, cigarette smoke–injured bronchial epithelial cells can survive proliferate as evidenced by clonogenic assay. These results suggest that DNA repair processes can be initiated after DNA damage by cigarette smoke.

DNA repair largely depends on enzymes such as DNA-dependent protein kinase (DNA-PK) and PARP (23, 24). PARP is activated in response to DNA damage or inactivated through cleavage by proteases such as caspase 3, resulting in failure of DNA repair (24). The current study found that cigarette smoke increased intact PARP synthesis in a concentration-dependent manner, but did not cleave the intact PARP into its inactive forms. Consistently, cigarette smoke increased caspase 3 precursor synthesis, but did not trigger the cleavage of caspase 3 into its active form. Furthermore, cigarette smoke did not increase caspase 3 activity, as determined by functional assay, suggesting that caspase 3 may not be involved in the process of cigarette smoke–induced DNA damage. In contrast, CPT exposure did activate caspase 3 as well as cleave intact PARP into its smaller molecular forms. In addition, the PARP inhibitor 3-ABA blocked DNA repair following cigarette smoke–induced DNA damage, as TUNEL positivity remained unchanged in the presence of 3-ABA after smoke was removed. Nevertheless, inhibition of PARP by 3-ABA in the presence of cigarette smoke did not increase apoptosis in that sub-G1 peak was not increased by 3-ABA plus cigarette smoke. Other studies also have reported increases in PARP following ultraviolet- or bleomycin-induced DNA breakage, and that the PARP inhibitor (3-ABA) decreased ultraviolet resistance in mammalian cells but increased the sensitivity to bleomycin-induced DNA breakage in mouse lung endothelial cells (25). These results suggest that PARP is activated in response to DNA damage induced by several types of insults, including cigarette smoke. The mechanism of 3-ABA blockade on DNA repair without inducing apoptosis, however, remains to be defined. It is possible that cells cultured beyond the 24-h time frames evaluated in the current study may have initiated apoptosis.

Several kinds of detection techniques for apoptosis have been developed, each with its advantages and limitations. The annexin-V affinity assay detects early apoptotic events, but is not suitable for attached cells because the enzymatic detaching of the cells may cause phosphatidylserine exposure at the outer cell membrane, thus potentially producing false-positive results. The DNA ladder on agarose gels can be used to detect DNA fragmentation during the later stages of apoptosis. This method, however, does not provide information regarding apoptosis in individual cells and does not detect ss-DNA break. In the last few years, techniques based on detection of DNA strand breaks have been widely used to identify cells undergoing apoptosis. These include TUNEL, DNA polymerase I–mediated in situ end-labeling (ISEL), Comet assay, and DNA-SB unwinding assay. TUNEL is more sensitive than ISEL in that TUNEL labels all DNA ends; that is, 5'-overhangs, 3'-overhangs, nick points, and blunt ends, whereas ISEL labels only 3'-overhangs. The principle of DNA-SB unwinding assay is that the rate of DNA unwinding in alkali depends on the length of ds-DNA, and that ethidium bromide binds selectively to ds-DNA in the presence of alkali. Thus, DNA-SB assay is used to assess ds-DNA damage. However, cells with positive TUNEL staining or ss-DNA damage may or may not proceed to apoptotic death. To identify genuine apoptotic cells, observation of characteristic cell morphologic changes under a microscope, detection of DNA fragmentation by DNA ladder and Comet assay, or detection of low molecular weight DNA by flow cytometry, also known as profiling of DNA content, can be performed. Comet assay evaluates apoptotic events of individual cells, whereas DNA ladder and DNA content profiling are used to evaluate cell population. Cells with fan-like tail and small heads in the Comet assay are considered as apoptotic cells (26, 27). The comet assay can also evaluate the intensity of DNA damage by computerized analysis of several comet parameters including Lhead, Ltail, head DNA, tail DNA, Lcomet, TM, and OTM (16, 26, 28). In the current study, TUNEL assay, DNA-SB assay, Comet assay, and profiling of DNA content were performed. Cigarette smoke significantly increased TUNEL positivity in bronchial epithelial cells and BEAS-2B cells, but did not decrease total ds-DNA percentage by DNA-SB assay, or increase percentage of apoptotic cells characterized with typical sub-G1 peak by DNA content profiling. In addition, compared with control cells, cigarette smoke–exposed cells had longer TM, OTM, and Lcomet, suggesting that DNA damage occurred in cigarette smoke–exposed cells. However, cigarette smoke did not increase the apoptotic index assessed by Comet, whereas CPT did, confirming the FACS data demonstrating that cigarette smoke did not induce apoptosis.

Clinically, consequences of abnormal regulation of apoptosis may be related to a variety of diseases, including lung cancer, emphysema, and fibrosis. Excessive apoptosis and insufficient recovery from DNA damage may lead to diseases such as emphysema, which is characterized by cell loss and connective tissue destruction. In contrast, failure of appropriate apoptotic cell death or aberrant DNA repair responses after exposure to cigarette smoke may leave bronchial epithelial cells with mutations that could contribute to the development of cancer. Furthermore, cells that undergo DNA damage and apoptotic pathway activation but survive may have altered function that could contribute to the pathogenesis of various lung diseases.


   Acknowledgments
 
The authors acknowledge the excellent secretarial support of Ms. Lillian Richards and the editorial assistance of Ms. Mary C. Tourek.


   Footnotes
 
The work was funded by the Larson Endowment, University of Nebraska Medical Center, Omaha, Nebraska.

Received in original form September 16, 2003

Received in final form April 14, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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